Silvia Ruiz-Gaspa

Effect of glucocorticoid treatment on Wnt signalling antagonists (sclerostin and Dkk-1) and their relationship with bone turnover

The aim of this study was to analyse the effect of glucocorticoid therapy (GCCT) on Wnt signalling antagonists (sclerostin and Dkk-1) and their relationship with bone turnover. 25 patients (8 M/17 F, aged 48±19yrs) recently initiating GCCT (≥7.5mg/day, ≤6months) were prospectively included. Bone turnover markers (bone formation: P1NP, osteocalcin [OC], bone ALP; bone resorption: sCTx) and Wnt antagonists (serum sclerostin and Dkk-1) were assessed in all patients (short-term and 12months after initiating GCCT). Bone mineral density (BMD) was performed to assess osteoporosis. The results were compared with 60 healthy controls. At short-term patients on GCCT showed a significant decrease in bone formation markers versus controls (P1NP: 19±9 vs. 43±16ng/mL, p<0.001; OC: 7.4±2.4 vs. 18.4±5.2ng/mL, p=0.001) and in Dkk-1 levels (24.5±20.1 vs. 36.8±13.7pmol/L, p=0.008) with similar sclerostin values (41.8±21.8 vs. 42.1±13.9pmol/L, p=0.950). Sclerostin correlated positively with GCCT doses (r=0.449, p=0.024) and lumbar BMD (r=0.424, p=0.035), and negatively with bone ALP (r=-0.398, p=0.049). A progressive decrease in Dkk-1 levels was observed at 12months, (19.1±14.9, p=0.001), whereas sclerostin increased compared to controls (48.9±11.6, p=0.045). In conclusion, the effect of GCCT on the serum levels of the Wnt signalling parameters differs depending on the antagonist evaluated. Whereas sclerostin values increased and showed a relationship with the dose and bone AP, Dkk-1 levels decreased throughout the study suggesting a counter-regulatory mechanism of this factor thereby reducing the deleterious effect of GCCT in the bone.

Lithocholic acid downregulates vitamin D effects in human osteoblasts.

Eur J Clin Invest 2010; 40 (1): 25–34

Effects of bilirubin and sera from jaundiced patients on osteoblasts: Contribution to the development of osteoporosis in liver diseases†

Low bone formation is considered to be the main feature in osteoporosis associated with cholestatic and end‐stage liver diseases, although the consequences of retained substances in chronic cholestasis on bone cells have scarcely been studied. Therefore, we analyzed the effects of bilirubin and serum from jaundiced patients on viability, differentiation, mineralization, and gene expression in the cells involved in bone formation. The experiments were performed in human primary osteoblasts and SAOS‐2 human osteosarcoma cells. Unconjugated bilirubin or serum from jaundiced patients resulted in a dose‐dependent decrease in osteoblast viability. Concentrations of bilirubin or jaundiced serum without effects on cell survival significantly diminished osteoblast differentiation. Mineralization was significantly reduced by exposure to 50 μM bilirubin at all time points (from −32% to −55%) and jaundiced sera resulted in a significant decrease on cell mineralization as well. Furthermore, bilirubin down‐regulated RUNX2 (runt‐related transcription factor 2) gene expression, a basic osteogenic factor involved in osteoblast differentiation, and serum from jaundiced patients significantly up‐regulated the RANKL/OPG (receptor activator of nuclear factor‐κB ligand/osteoprotegerin) gene expression ratio, a system closely involved in osteoblast‐induced osteoclastogenesis. Conclusion: Besides decreased cell viability, unconjugated bilirubin and serum from jaundiced patients led to defective consequences on osteoblasts. Moreover, jaundiced serum up‐regulates the system involved in osteoblast‐induced osteoclastogenesis. These results support the deleterious consequences of increased bilirubin in advanced chronic cholestasis and in end‐stage liver diseases, resulting in disturbed bone formation related to osteoblast dysfunction. (HEPATOLOGY 2011)

Ursodeoxycholic acid increases differentiation and mineralization and neutralizes the damaging effects of bilirubin on osteoblastic cells.

Osteoporosis resulting from decreased bone formation is a common complication in patients with chronic cholestasis. Lithocholic acid (LCA) and bilirubin may play a role in osteoporosis given that both substances have detrimental effects on survival of human osteoblasts, the cells involved in bone formation.

Sclerostin Expression in Bile Ducts of Patients With Chronic Cholestasis May Influence the Bone Disease in Primary Biliary Cirrhosis

Sclerostin is involved in the regulation of osteoblastogenesis and little is known about its role in the development of bone disease in primary biliary cirrhosis (PBC), characterized by low bone formation. Therefore, we have assessed the circulating levels and the liver expression of sclerostin in this cholestatic disease. Serum sclerostin levels were measured in 79 women with PBC (mean age 60.6 ± 1.2 years) and in 80 control women. Lumbar and femoral bone mineral density (BMD), as well as parameters of mineral metabolism and bone remodeling, were measured. Moreover, sclerostin gene (SOST) expression in the liver was assessed by real‐time PCR in samples of liver tissue taken by biopsy in 11 PBC patients and in 5 normal liver specimens. Presence and distribution of sclerostin was evaluated in liver slices from 11 patients by immunohistochemistry. The severity of histologic lesions was assessed semiquantitatively in the same liver samples. PBC patients had higher sclerostin levels than controls (75.6 ± 3.9 versus 31.7 ± 1.6 pmol/L, p < 0.001). Serum sclerostin correlated inversely with markers of bone formation and resorption. Sclerostin mRNA in the liver was overexpressed compared with control samples (2.7‐fold versus healthy liver). Sclerostin was detected by immunohistochemistry in 7 of the 11 liver samples, mainly located in the bile ducts. Liver sclerostin was associated with the severity of cholangitis (p = 0.02) and indirectly with the degree of lobular inflammation (p = 0.03). Sclerostin mRNA expression was higher in samples that tested positive by immunohistochemistry and particularly in those with lobular granuloma (p = 0.02). The increased expression of sclerostin in the liver and the association with histologic cholangitis may explain the high serum levels of this protein in patients with PBC, thus suggesting that sclerostin may influence the decreased bone formation in this cholestatic disease.

Effect of Recent Spinal Cord Injury on Wnt Signaling Antagonists (Sclerostin and Dkk‐1) and Their Relationship With Bone Loss. A 12‐Month Prospective Study

Spinal cord injury (SCI) has been associated with a marked increase in bone loss and bone remodeling, especially short‐term after injury. The absence of mechanical load, mediated by osteocyte mechanosensory function, seems to be a causative factor related to bone loss in this condition. However, the pathogenesis and clinical management of this process remain unclear. Therefore, the aim of the study was to analyze the effect of recent SCI on the Wnt pathway antagonists, sclerostin and Dickkopf (Dkk‐1), and their relationship with bone turnover and bone mineral density (BMD) evolution. Forty‐two patients (aged 35 ± 14yrs) with a recent (<6months) complete SCI were prospectively included. Sclerostin and Dkk‐1, bone turnover markers (bone formation: PINP, bone ALP; resorption: sCTx) and BMD (lumbar spine, proximal femur, total body and lower extremities [DXA]) were assessed at baseline and at 6 and 12 months. The results were compared with a healthy control group. 22/42 patients completed the 12‐month follow‐up. At baseline, SCI patients showed a marked increase in bone markers (PINP and sCTx), remaining significantly increased at up to 6 months of follow‐up. Additionally, they presented significantly increased Dkk‐1 values throughout the study, whereas sclerostin values did not significantly change. BMD markedly decreased at the proximal femur (‐20.2 ± 5.4%, p < 0.01), total body (‐5.7 ± 2.2%, p = 0.02) and lower extremities (‐13.1 ± 4.5%, p = 0.01) at 12 months. Consequently, 59% of patients developed densitometric osteoporosis at 12 months. Patients with higher Dkk‐1 values (>58 pmol/L) at baseline showed higher sublesional BMD loss. In conclusion, this study shows that short‐term after SCI there is a marked increase in bone turnover and bone loss, the latter associated with an increase in Dkk‐1 serum levels. The persistence of increased levels of this Wnt antagonist throughout the study and their relationship with the magnitude of bone loss suggests a contributory role of this mediator in this process.

Ursodeoxycholic acid decreases bilirubin-induced osteoblast apoptosis.

Low bone turnover osteoporosis is common in cholestatic diseases. Ursodeoxycholic acid (UDCA) counteracts the damaging effects of bilirubin or lithocholic acid (LCA) on osteoblast viability, proliferation and mineralisation. UDCA is anti‐apoptotic in various cell lines, but this effect in bone cells is unknown. Therefore, the consequences of bilirubin and LCA on apoptosis, and whether UDCA has anti‐apoptotic effects have been assessed on osteoblasts.

Idiopathic Acquired Osteosclerosis in a Middle-Aged Woman With Systemic Lupus Erythematosus

Widely distributed osteosclerosis is an unusual radiographic finding with multiple causes. A 42‐year‐old premenopausal Spanish woman gradually acquired dense bone diffusely affecting her axial skeleton and focally affecting her proximal long bones. Systemic lupus erythematosus (SLE) diagnosed in adolescence had been well controlled. She had not fractured or received antiresorptive therapy, and she was hepatitis C virus antibody negative. Family members had low bone mass. Lumbar spine bone mineral density (BMD) measured by dual‐photon absorptiometry (DPA) at age 17 years, while receiving glucocorticoids, was 79% the average value of age‐matched controls. From ages 30 to 37 years, dual‐energy X‐ray absorptiometry (DXA) BMD Z‐scores steadily increased in her lumbar spine from +3.8 to +7.9, and in her femoral neck from –1.4 to –0.7. Serum calcium and phosphorus levels were consistently normal, 25‐hydroxyvitamin D (25OHD) <20 ng/mL, and parathyroid hormone (PTH) sometimes slightly increased. Her reduced estimated glomerular filtration rate (eGFR) was 38 to 55 mL/min. Hypocalciuria likely reflected positive mineral balance. During increasing BMD, turnover markers (serum bone‐specific alkaline phosphatase [ALP], procollagen type 1 N propeptide [P1NP], osteocalcin [OCN], and carboxy‐terminal cross‐linking telopeptide of type 1 collagen [CTx], and urinary amino‐terminal cross‐linking telopeptide of type 1 collagen [NTx and CTx]) were 1.6‐ to 2.8‐fold above the reference limits. Those of bone formation seemed increased more than those of resorption. FGF‐23 was slightly elevated, perhaps from kidney disease. Serum osteoprotegerin (OPG) and TGFβ1 levels were normal, but sclerostin (SOST) and receptor activator of nuclear factor kappa‐B ligand (RANKL) were elevated. Serum multiplex biomarker profiling confirmed a high level of SOST and RANKL, whereas Dickkopf‐1 (DKK‐1) seemed low. Matrix metalloproteinases‐3 (MMP‐3) and ‐7 (MMP‐7) were elevated. Iliac crest biopsy revealed tetracycline labels, no distinction between thick trabeculae and cortical bone, absence of peritrabecular fibrosis, few osteoclasts, and no mastocytosis. Then, for the past 3 years, BMD Z‐scores steadily decreased. Skeletal fluorosis, mastocytosis, myelofibrosis, hepatitis C‐associated osteosclerosis, multiple myeloma, and aberrant phosphate homeostasis did not explain her osteosclerosis. Mutation analysis of the LRP5, LRP4, SOST, and osteopetrosis genes was negative. Microarray showed no notable copy number variation. Perhaps her osteosclerosis reflected an interval of autoimmune‐mediated resistance to SOST and/or RANKL.

1306 URSODEOXYCHOLIC ACID NEUTRALIZES THE NOXIOUS EFFECTS OF LITHOCHOLIC ACID AND BILIRUBIN ON OSTEOBLASTS

did not differ between the two groups. ALT response was inversely correlated with HA, BDL and F score, while ALP response was inversely correlated with BDL score. In addition, positive anti-gp210 antibodies was significantly associated with poor response of both ALT and ALP. Conclusions: Positive anti-gp210 antibodies is a significant risk factor for poor biochemical response to UDCA treatment in Japanese patients with PBC.